2002 Traffic

Copyright C Blackwell Munksgaard 2002Traffic 2002; 3: 621–629

Blackwell Munksgaard ISSN 1398-9219

Review

Membrane Trafficking During Plant Cytokinesis

Sebastian Y. Bednarek* and Tanya G. Falbel

Department of Biochemistry, University of Wisconsin-

Madison, 433 Babcock Dr, Madison WI 53706, USA

* Corresponding author: Sebastian Y. Bednarek,

[email protected]

Plant morphogenesis is regulated by cell division andexpansion. Cytokinesis, the final stage of cell division,culminates in the construction of the cell plate, aunique cytokinetic membranous organelle that is as-sembled across the inside of the dividing cell. Both dur-ing cell-plate formation and cell expansion, the se-cretory pathway is highly active and is polarized to-ward the plane of division or toward the plasmamembrane, respectively. In this review, we discuss re-sults from recent genetic and biochemical research di-rected toward understanding the molecular events oc-curring during cytokinesis and cell expansion, includ-ing data supporting the idea that during cytokinesisone or more exocytic pathways are polarized towardthe division plane. We will also highlight recent evi-dence for the roles of secretory vesicle transport andcytoskeletal machinery in cell-plate membrane traf-ficking and fusion.

Key words: cell division, cell plate, cytoskeleton, dyna-min, exocytosis, Rop, SNARE

Received 14 June 2002, revised and accepted for pub-lication 17 June 2002

Cytokinesis, the grand finale of a cell division, involves the par-titioning of cytosol and organelles and the completion of amembranous barrier between daughter cells. Formation of thismembranous barrier in higher plant cells involves the de novo

assembly of the unique cytokinetic organelle, the cell plate, aprocess that involves polarized trafficking of massive amountsof membrane and cell-wall material into the division plane. Theextent of this flow of membrane is dramatically illustrated in di-viding cambial initials during wood development where the cellplate is several millimeters long and requires approximately aday to complete (see Figure 1). Cell-plate assembly is orches-trated by the phragmoplast, a specialized cytoskeletal struc-ture, composed of microtubules (MTs) and microfilaments(MFs) (1). During cytokinesis, secretory vesicles carrying mem-brane and cell-wall components are guided along the phrag-moplast toward its center, where they fuse to form a membra-nous tubular-vesicular network (TVN) (2), within which cell-wall biosynthesis is initiated. As additional vesicles are added,the TVN gradually develops into a smoother, more plate-like

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structure that expands outward toward the margin of the cell.Ultimately the cell plate fuses with the parental plasma mem-brane at a site coincident with the previous location of the pre-prophase band, a transient ring of cortical MTs that formsbriefly during the G2 phase of the cell cycle. Alignment of theexpanding cell plate with the fusion site is regulated by an acto-myosin system (3–5) which appears to push the cell plate out-ward. Conversely, in animal cells, a contractile actomyosin ringbrings the plasma membrane inward. Recent studies in animalcells and fungi (6,7) now demonstrate the importance of tar-geted secretion of membranes for furrowing and resolution ofthe constricted plasma membrane [for review, see (8–10].Therefore, although the mechanism for cytokinesis in plantsand fungi and animals has long been considered wholly dis-tinct, these systems in fact bear significant similarities.

In this review we will focus primarily on recent studies ofmembrane trafficking during plant cytokinesis, making com-parisons with other systems where possible, as well as high-lighting significant remaining questions about the molecularmechanisms involved in cell-plate membrane trafficking andfusion. For additional discussion of the various topics touchedupon here, the reader is referred to several other recent re-views (11–15).

Organization of the Secretory MachineryDuring Cytokinesis

Differences exist in the organization of the secretory path-way between plant and animal cells. Most notably, plantcells contain multiple independent Golgi stacks (i.e. oftenhundreds) that do not vesiculate during mitosis as is thecase in animal cells. This is important because the processof secretion in plants remains active throughout the cellcycle. Plant Golgi stacks are highly mobile, moving activelythroughout the cytoplasm along actin filaments via an acto-myosin system (16–18). During mitosis these stacks ac-cumulate in a subcortical ring, the so-called ‘Golgi Belt’,surrounding the future site of cell-plate formation (17). Thislocalization pattern does not depend upon cytoskeletal in-teractions, but is thought to be required for partitioning ofthe Golgi into the daughter cells as well as for delivery ofsecretory vesicles to the cell plate. During mitosis andcytokinesis, tubular components of the endoplasmic retic-ulum (ER) are also recruited to the division plane, as shownby electron microscopy (19,20) and three-dimensional con-focal imaging (21) to form a reticular network surroundingthe cell plate. Analogous to the cortical ER, which is ad-

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Figure1: Ultrastructure of a dividing vascular cambial cell in developing wood tissue of Pine. The cell plates of these dividingcambial initials can be several millimeters long. An immature region of this cell plate is shown at the top part of the figure and in the inset.Secretory vesicles are seen starting to coalesce into a tubulo-vesicular network. The lower part of the figure shows an older region of thecell plate where the membrane has flattened into a more plate-like structure that will further mature and thicken as cellulose biosynthesisbegins to form the new cell wall. PCW: parental cell wall. G: Golgi stack. Tissue samples were preserved by high-pressure freezing andfreeze substitution (85). Image courtesy of Drs Lacey Samuels and Kim Rensing (University of British Columbia).

jacent to the plasma membrane, the cell-plate-associatedER may mediate direct lipid transfer to the cell plate and/or provide the appropriate ionic environment (e.g. supplyingCa2π ions) necessary for cell-plate membrane fusion andcytoskeletal organization. Tubular elements of the cell-plate-associated ER network may also fuse and becomeentrapped, forming the desmotubules that traverseplasmodesmata. How these membranes are recruited tothe division plane and assembled remains to be deter-mined.

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Multiple Secretory Routes May Lead to theParental Plasma Membrane and Cell Plate

Plant cell expansion is mediated through the delivery and fu-sion of Golgi-derived exocytic vesicles carrying membranesand cell-wall components to specific sites on the plasmamembrane. Likewise, the vesicles that fuse at the site of cell-plate formation are thought to bud from the Golgi apparatus.However, very little is known about the molecular machineryinvolved in their formation and targeting in plant cells. For


example, it remains to be determined whether cell-plate for-mation is mediated by a single homogeneous vesicle popula-tion or if multiple Golgi-derived vesicle types, carrying distinctsets of cargo, converge and fuse to form a new plasma mem-brane and cell wall de novo. Recent studies have demon-strated the existence of multiple exocytic pathways thattransport distinct sets of cargo to the cell surface in yeast andmammalian cells (22–24). Similarly the presence of multipleexocytic pathways in plant cells (Figure 2A) has been sug-gested, based upon the polarized distribution of Golgi-syn-thesized polysaccharides in the cell walls of root tip and sus-pension-cultured cells (25,26). The recent localization and

Figure2: Speculative model for polarized vesicular trafficking during (A) polarized cell-surface expansion and (B) cell-plateformation. A: In polarized growth, vesicles are delivered by multiple exocytic pathways (open and closed red circles) toward sites ofexpansion on the plasma membrane (red). Targeting of these vesicles may be guided by short MFs (black). B: During cytokinesis, exocyticvesicles are directed toward the cell plate (red). Targeting of these vesicles is guided by phragmoplast MTs (green) and short MFs (black).Cell-plate transport vesicles may originate directly from the Golgi apparatus or from an intermediate compartment (e.g. an early and/or lateendocytic/prevacuolar endocytic compartment). Membrane is recycled via clathrin-coated vesicles (white hexagons) from the plasma mem-brane and cell plate (blue arrows) and delivered to an endocytic compartment (pink). See text for additional details. CP: cell plate, E: endocyticcompartment, G: Golgi, N: nuclei, V: vacuole. The ER is not shown.

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characterization of the polarized distribution of several cell-wall-associated and plasma membrane proteins, includingCOBRA (27) and the putative auxin transporters PIN1, PIN3and AUX1 (28–30), lends further support to this model [fordiscussion, see the article by Jürgens and Geldner in thisissue (31)].

During cell-plate formation, one or more of these pathwaysmay become polarized toward the plane of division, as theyappear to do during cell division in Saccharomyces cerevis-

iae (32,33). Alternatively, it has been suggested, based uponthe localization of the cell-plate SNARE, KNOLLE, that a

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unique class of cell-plate-specific transport vesicle(s) may begenerated during cytokinesis (34). KNOLLE was found to beexpressed exclusively in dividing cells and targeted specifi-cally to the cell plate during cytokinesis. More recent studiesshow several constitutively synthesized proteins, includingsoluble endoxyloglucan transferase (35), and the plasma-membrane-associated proteins KORRIGAN (36), PIN1 (37)and plasma membrane PM-ATPase (Dickey and Bednarek,unpublished) are localized at the cell plate during cell division.These data argue in favor of a model in which the exocyticsecretory pathway becomes polarized toward the divisionplane during cytokinesis, and suggests that it is the cell-cycletiming of secretory protein synthesis that dictates whetherthey are transported to the cell surface or the cell plate. Insupport of this proposal, constitutive expression of KNOLLEresulted in its localization to the plasma membrane in nondi-viding cells and to the cell plate in dividing cells (38). Severalquestions that remain to be addressed are whether the exo-cytosis of plasma membrane and extracellular proteins to theparental cell surface continues during cytokinesis, andwhether one or more exocytic pathways are directed towardthe cell plate. With regard to the latter, it will be interestingto determine if in addition to PIN1 other asymmetrically dis-tributed plasma membrane proteins such as AUX1 (30) arealso targeted to the cell plate.

Sorting signals

Polarized trafficking of KORRIGAN, an endo-1,4-beta-glucan-ase required for cell expansion and cytokinesis, has beenshown to depend on two sorting signals within the cytosolicN-terminal domain of the protein (36). Mutation of either oneof these signals, an acidic di-leucine (LL) motif [typically apair of leucines located 4–5 amino acids C-terminal of anacidic residue (39)] or a YXXj [Y is tyrosine, X is any aminoacid and j a hydrophobic amino acid with a large side chain(40)] resulted in the persistent mislocalization of KORRIGANat the plasma membrane. Both of these sorting motifs arealso present in the cytosolic domains of PIN1, KNOLLE, andanother cell-plate-associated SNARE NSPN11 (41), but theirrole in trafficking to the cell plate remains to be defined.Studies in yeast and mammalian cells have demonstrate thatthe YXXj and di-LL motifs are recognized by GGAs (Golgi-localized g-ear-containing ARF binding proteins) and the ad-aptor protein complexes AP-1, AP-2, AP-3 [for review, see(42) and references therein] which are responsible for traf-ficking of cargo proteins via clathrin-coated vesicles betweenthe TGN, plasma membrane and endocytic compartments.Similarly a YXXj motif in the C-terminus of the putative plantvacuolar cargo receptor that traffics between the TGN and aprevacuolar compartment has been shown to interact withthe AP-1 complex (43). Interestingly, the asymmetric plasmamembrane distribution of PIN1 involves the cycling of theprotein between the plasma membrane and an endosomalcompartment (38). KNOLLE and NSPN11 have also been ob-served in punctate subcellular organelles resembling endo-somes in dividing and nondividing Arabidopsis cells (41).These observations suggest the intriguing possibility that thepolarized trafficking of proteins containing the acidic-LL and

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YXXj sorting motifs to the cell plate may involve transportthrough the endocytic pathway (see Figure 2B). Consistentwith this hypothesis, one of two exocytic pathways from theTGN to the plasma membrane in yeast has recently beenshown to involve an intermediate endocytic compartment,the prevacuolar compartment (24). Whether a similar mech-anism exists in plants and whether the acidic-LL and YXXj

sorting signals mediate the general sorting of transmem-brane proteins including KNOLLE, NSPN11, and PIN1 to thecell plate during cytokinesis remains to be determined.

Cell-Plate Secretory Trafficking and FusionMachinery

The formation, targeting and fusion of transport vesicleswithin each branch of the plant exocytic and endocytic se-cretory pathway is assumed to be regulated by a wide varietyof cytosolic and membrane-associated factors that are highlyconserved among all eukaryotic organisms [for review, see(44)].

Dynamins

Formation of transport vesicles involves the assembly of dis-tinct coat complexes that drive membrane budding and theselection of cargo proteins. This process is regulated by smallGTPases such as ARF (ADP-Ribosylation Factor) that are re-quired for the formation of TGN-derived clathrin coated ves-icles. In mammalian cells, release of the clathrin-coated TGN-derived and endocytic vesicles from the plasma membraneinvolves the action of another GTPase, dynamin, the definingmember of structurally related but functionally diverse familyof large GTP-binding proteins (45).

Dynamin has been shown to form multimeric rings aroundthe necks of invaginating clathrin-coated vesicles and othermembrane tubules. In the presence of GTP these dynamin-coated membranes have been observed to fragment, sug-gesting that the dynamin rings function as a macromoleculargarrote. An alternative but not mutually exclusive idea is thatthe dynamin rings serve to recruit various binding partners,such as the lipid modification enzyme endophilin and othervesicle budding components that function in concert with dy-namin to promote membrane scission (46). In addition totheir role in vesicular trafficking, dynamin and dynamin-re-lated proteins have been linked to a number of diverse pro-cesses including actin dynamics (47).

The plant-specific, dynamin-related protein from soybean,phragmoplastin (48), and its Arabidopsis homologue, the Ar-abidopsis dynamin-like protein (ADL1) (34,40–51), have beenlocalized to the cell plate. The ADL1 protein family has fivemembers which are�80% similar in sequence. Like dynam-in, phragmoplastin and ADL1A have been shown to formhelical structures both in vitro (52) and in vivo on syncytial-type cell plates during the endosperm cellularization in devel-oping Arabidopsis seeds (51). ADL1A rings were found at�20nm constrictions throughout the initial wide tubular


membrane system of the developing syncytial-type cellplates. In contrast to dynamin, these rings do not appear tosever the membrane tubules, suggesting that they may berequired to maintain the tubular nature of the initial syncytialcell plate prior to its consolidation (51). Similar to its proposedrole in syncytial-type cell-plate development, ADL1A andphragmoplastin have been suggested to play a role in theformation/stabilization of �20nm diameter fusion tubulesthat have been observed to connect fusing cell-plate vesicles(2,15). However, the latter association of these dynamin-likeproteins to these tubules remains to be confirmed by immu-no-electron microscopy (EM).

Analysis of adl1A loss-of-function mutants has confirmedthat ADL1A is required for various stages of plant growth anddevelopment; however, the mutants do not display defects incytokinesis (50). One explanation for this is that functionallyredundant ADL1 proteins may compensate for the loss ofADL1A during cell division. Indeed, other members of theADL1 gene family display overlapping expression patternsthroughout plant development, and moreover colocalize withADL1A at the cell plate during cytokinesis (Kang and Bedna-rek, unpublished). More recent studies have suggested a rolefor ADL1A in polarized vesicular trafficking and endocytosis.ADL1A and other proteins involved in vesicular transport be-tween the TGN, plasma membrane and endocytic compart-ments including b-adaptin, a subunit of the clathrin adaptorprotein complex, and AtSEC14 cofractionated by affinitychromatography on immobilized-naphthylphthalmic acid(NPA) (53). NPA is an inhibitor of polar auxin transport thathas been shown to block trafficking of PIN1, KNOLLE, andthe PM-ATPase (37). Consistent with this, adl1A mutants dis-play cell-type specific defects in the polarized growth andendocytosis (Kang and Bednarek, unpublished). These re-sults suggest that during cytokinesis ADL1A may function inthe formation of cell-plate transport vesicles and in the recyc-ling of membranes and vesicles from the developing cellplate. Nearly 75% of the total membrane delivered to the cellplate has been estimated to be removed during cell-platematuration (51) indicating that regulation of membrane recyc-ling is likely to be critical for cytokinesis. Other additional plantdynamin-related proteins, including the mammalian dynaminhomolog ADL6 (54), have been localized to the cell plate (15)and may be required for cell-plate formation.

In addition to their potential roles in cell-plate membrane traf-ficking, ADL1A and phragmoplastin may also regulate 1,3-b-glucan (callose) biosynthesis (15). Callose deposition withinthe cell-plate lumen precedes cellulose synthesis and isthought to help drive the spreading and stabilization of cell-plate membranes (1). The catalytic subunit of callose syn-thase has been localized to the cell plate (55) and shownto interact directly with phragmoplastin. Overexpression ofphragmoplastin has also been found to stimulate the ac-cumulation of callose (15), suggesting that it may regulatethe activity or localization of the callose synthase complex.Interestingly, putative acidic LL and YXXj sorting motifs arepresent in the N-terminal cytosolic domain of callose syn-

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thase, which could facilitate its targeting to the cell plate andplasma membrane.

GNOM and cell-plate formation

With the possible exception of the above-mentioned dynam-in-like proteins, no other factors involved in the biogenesis oftransport vesicles that are directed to the cell plate have beenidentified to date. GNOM/EMB30 is an ADP-ribosylation fac-tor-guanine nucleotide exchange factor (ARF-GEF) that is re-quired for the polarized transport of PIN1 to the plasmamembrane (56). Yet, surprisingly, gnom/emb30 mutantsshow no obvious cytokinesis defects. One explanation for thisis that disruption of GNOM/EMB30 may only affect one ofseveral exocytic pathways that are directed to the cell plate(Figure 2) and that loss of a single vesicular transport path-way to the cell plate could be compensated for by the othercell-plate-directed exocytic pathways. Similarly, yeast mu-tants lacking one of the two exocytic branches are viablebecause the remaining functional pathway to the cell surfacebegins to transport the cargo proteins of the other pathway(24).

Rabs and tethering systems

Following secretory vesicle formation, another class of smallGTPases, Rabs, function to coordinate the docking (alsoknown as tethering) of transport vesicles to their appropriateacceptor membranes (57). Each branch of the secretorypathway is thought to contain a unique Rab that recruits alarge fibrous protein or protein complex, a vesicle-tetheringfactor, to sites where transport vesicle and their target mem-brane fuse. The yeast genome encodes only 11 Rabs,whereas animals and plants have over 50 distinct Rabs (58).This significant difference may be related to specific se-cretory needs of complex multicellular organisms, includingmultiple polarized exocytic pathways. With the exception ofRab1 and Rab2, which function in ER-to-Golgi trafficking(18,59), little is known about the function of other plant Rabs.The roles of other plant Rabs have only been inferred basedupon sequence homology and localization studies. Recently,Rab11 (which regulates trafficking between the TGN, endo-somes and plasma membrane domains) was demonstratedto be required for the completion of cytokinesis in early Ca-

enorhabditis elegans embryos (60). The importance of Rabsfor cell-plate formation is supported by the recent identifi-cation of SCD1 (stomatal cytokinesis defective), a gene thatis required for cytokinesis in developing stomates and theexpansion of various other cell types (Falbel et al., unpub-lished data). SCD1 encodes a protein homologous to guaninenucleotide exchange factors for Rab GTPases, putatively re-presenting the first regulatory link to Rab GTPases andtethering systems in the pathway of cell-plate formation.

Compared to plant Rabs, even less is known about the exist-ence and function of plant secretory vesicle-tethering factors.The Arabidopsis genome appears to encode homologs ofsome subunits of the S. cerevisiae exocyst complex, aneight-subunit plasma membrane-associated tethering com-plex required for exocytosis (57). However, no functional

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analysis has been performed yet for the putative Arabidopsisexocyst or other tethering complexes.

Cell-plate membrane fusion factors

Following tethering it has been proposed that pairing of cog-nate SNAREs, that reside on the opposing membranes (i.e.vesicle v-SNAREs pair with target membrane t-SNAREs)drives transport vesicle fusion. The post-fusion or cis-SNAREcomplex is comprised of a highly stable parallel-four helixbundle in which one of the helices is contributed from a v-SNARE, one from a syntaxin-type t-SNARE, and two fromeither a single SNAP-25-type SNARE or two other individualt-SNAREs [for review, see (44)]. The recent identification andcharacterization of several Arabidopsis membrane-fusion fac-tors that localize to the division plane have provided someinsight into the membrane-fusion mechanism(s) required forplant cytokinesis.

KNOLLE, the cell-plate-associated t-SNARE (34,61), hasbeen shown to bind to SNAP33 (a SNAP25-type SNARE)(62), the plant-specific SNARE, NSPN11 (41) and the Sec1phomolog, KEULE (63). Sec1 family members exhibit speci-ficity for particular syntaxins. In concert with Rabs and ves-icle-tethering factors, they regulate syntaxin’s ability to inter-act with other SNAREs (57). Consistent with this, cells of se-verely malformed knolle and keule mutant Arabidopsis

seedlings display defects in cell-plate assembly and knolle/

keule double mutants are synthetically lethal (63,64), sug-gesting that these two genes are part of the same fusionpathway. It has been proposed that the interaction of KEULEand KNOLLE on the target membrane could prime a cell-plate membrane domain for the addition of the next vesicle(13,63). In contrast to knolle and keule, snap33 and nspn11

mutants show either very mild or no obvious cytokinesis de-fects (41,62). However, keule/snap33 and knolle/snap33

double mutants are synthetically lethal, providing genetic evi-dence that SNAP33 functions along with KNOLLE and KEU-LE during cell-plate–membrane fusion. Analysis of nspn11/

keule and nspn11/knolle double mutants remains to be done.One possible explanation for the phenotypes of the snap33

and nspn11 mutants is that SNAP33 and NSPN11 are mem-bers of multigene families (65) and thus mutations in eitherSNARE may be suppressed by expression of other familymembers. Indeed, KNOLLE interacts in a yeast two-hybridassay with two other Arabidopsis SNAP25-type SNAREs,SNAP29 and SNAP30 (62); however, the expression andlocalization of these SNAREs remains to be determined. Un-like KNOLLE, which is expressed only in dividing cells,SNAP33, NSPN11 and KEULE are expressed in dividing aswell as nondividing cells, suggesting that these proteins func-tion not only in cell-plate formation but also in other exocyticvesicle fusion events. In support, SNAP33 has been demon-strated to interact with the plasma membrane syntaxin SYR1(SYP121) (66).

SNARE complexes are enzymatically disassembled by theAAA (ATPases associated with various cellular activities) (67)chaperone NSF prior to or concomitant with tethering in or-

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der to prime the individual SNAREs for fusion (44). A secondAAA ATPase, CDC48 (p97 in mammalian cells) has also beenshown to interact with SNAREs during the homotypic fusionof ER-ER and transitional ER membranes (68,69). Interest-ingly, Cdc48p (p97) and NSF along with the SNARE, syntaxin5 mediate the fusion of mitotic Golgi membranes in mam-malian cells through two separate pathways. Likewise, someor all of the stages of ER and cell-plate membrane dynamicsat the division plane, including cell-plate vesicle fusion andconsolidation of the TVN into the smooth plate, may involveNSF- and/or Cdc48p-dependent mechanisms. Indeed,KNOLLE has been shown to interact with NSF in vitro (Ran-cour et al., unpublished data). Furthermore, the plant orthol-og of CDC48, AtCDC48, is localized at the division plane individing Arabidopsis cells (70). In vitro binding studies, how-ever, reveal that AtCDC48 does not interact with KNOLLE butrather with another division plane localized SNARE, SYP31,the plant ortholog of the ER-to-Golgi syntaxin 5 (Rancouret al., manuscript submitted). Although the significance ofAtCDC48 and SYP31 localization at the division plane re-mains to be determined, these results suggest that there areat least two distinct membrane fusion pathways involvingCDC48p/p97 and Sec18p/NSF that operate at the divisionplane to mediate plant cytokinesis.

Cytoskeletal Guidance of Cell-PlateMembrane Trafficking

The phragmoplast guides secretory vesicle trafficking to thedivision plane and the expansion of the developing cell plateoutward toward the cell cortex. This cytoskeletal scaffold ismade up of two antiparallel arrays of MTs and MFs, with theMT and MF plus ends directed toward the central equatorialregion of the structure. In animal cells, there appear to befunctional equivalents of the phragmoplast; the spindle mid-zone (60,71) and in some cell types the furrow microtubulearray (10) direct the insertion of membrane into the cleavagefurrow and are required for proper cytokinesis. Like thephragmoplast, each of these structures is composed of twoarrays of MTs with plus ends directed toward the center. Cell-plate formation is blocked by MT depolymerization and inmutants that disrupt tubulin folding (72). Similarly, MT de-polymerization in animal cells prevents the addition of newmembrane in the furrow and results in furrow regression (10).Thus, the arrival of membrane via a MT scaffold appears tobe a plant feature that is conserved in animals to accomplishcytokinesis.

Microtubule-associated motors

Plus end-directed MT motor proteins are thought to drive ves-icles toward the equatorial plane of the phragmoplast duringcell-plate formation. Ultrastructural analysis of developing cellplates has revealed that phragmoplast MT-associated trans-port vesicles are connected to the MT via rod-like 10–30nmstructures that resemble kinesin-type motor proteins (51). At-PAKRP2 (73) is a recently identified plus end-directed kines-in-like protein (KLP) that may function in vesicular transport


during cytokinesis. AtPAKRP2 is localized normally to phrag-moplast MTs, but the ARF-GEF inhibitor, Brefeldin A, whichblocks secretory vesicle trafficking, disrupts this localizationin dividing cells (73). AtPAKRP2 does not, however, colocalizewith KNOLLE, a marker of cell-plate transport vesicles. There-fore additional functional and immuno-EM studies arenecessary in order to establish the role of AtPAKRP2 in cell-plate vesicle trafficking.

Several other plus end-directed KLPs, such as TKRP125,NACK1/Hinkel, NACK2 and KCBP have been found to be as-sociated with the phragmoplast. However, they appear toplay a role in the assembly and maintenance of the phragmo-plast MT organization [for review, see (74,75)] and in phrag-moplast-guided lateral expansion of the cell plate (76,77), butnot in vesicle trafficking to the cell plate.

Role for the actin cytoskeleton in cell-plate vesicle

targeting and fusion?

In contrast to the MT-dependent trafficking of vesicles to thecell plate during cytokinesis, vesicle trafficking to the cell sur-face continues in the absence of MTs (72). Conversely,studies with actin-disrupting drugs have suggested a role forMFs in vesicular trafficking to the plasma membrane but notto the cell plate (5,37).

Two types of MFs have been observed in tip-growing cellssuch as pollen tubes: large actin cables, which are thoughtto mediate long-distance transport of secretory vesicles; andshort MFs, which are localized at the tip where the vesiclesfuse (78). The role for the short MFs is not understood; how-ever, they may serve as a landmark for targeting and fusionof the exocytic vesicles (Figure 2). Recent studies suggestthat members of the plant-specific Rho GTPase subfamily,Rops (Rho related GTPase of plants), regulate polarized cellexpansion by modulating the formation of short MFs at thecell cortex (78–80). Overexpression of constitutively activeand dominant-negative Rops resulted in the disassembly ormislocalization of short cortical MFs, respectively, both caus-ing defects in cell expansion (78). Consistent with their rolein polarized membrane expansion, Rops have been localizedto the apical plasma membrane of tip-growing cells and tosites on the surface of cells undergoing localized expansion.

During cytokinesis, Rops and short MFs are associated withthe cell plate and may function in its assembly. Similar to theshort cortical MFs involved in cell expansion, short MFs areassociated with the leading edge of the cell plate (81), whichis the predominant site where new membrane is incorpor-ated into the expanding cell plate. A green fluorescent proteintagged-Rop, GFP-AtRop4, was shown to be localized to thecell plate in transgenic tobacco BY2 cells (80) potentially di-recting the assembly of the short MFs. In addition, theRop1GTPase was shown to interact in its GTP-bound statewith a UDP-glucose transferase, a subunit of the cell-plate-associated phragmoplastin/callose synthase complex (82),suggesting that it may regulate callose biosynthesis. Alterna-tively, the interaction between Rop and the callose synthase

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complex may serve to coordinate membrane deposition andlocalized callose biosynthesis during cell-plate assembly.

Recent studies in S. cerevisiae have demonstrated that RhoGTPases, MFs, and Rab GTPases are all required for the spa-tial regulation of the vesicle tethering complex, the exocyst,thereby regulating the site at which vesicles fuse with theplasma membrane (83,84). In the fission yeast, Schizo-sac-

charomyces pombe, exocyst proteins localize in an F-actin-dependent manner to regions of active exocytosis, includingthe growing ends of interphase cells and medial regions ofcells undergoing cytokinesis (7). During cytokinesis in S.

pombe, mutants lacking the exocyst subunit Sec8p accumu-late unfused vesicles in the cytoplasm near the division sep-tum, which morphologically resembles the build-up of un-fused cell-plate vesicles in knolle mutant cells (34). Furthergenetic and biochemical experiments will help to establishwhether Rops and short MFs regulate the polarized targetingand fusion of transport vesicles at the cell plate and facilitatethe identification of other components of the cell-plate ves-icle targeting machinery, including the necessary Rabs andtethering factors.

Final Perspective

It has only been a few short years since the first high-resolutionimages of the dynamic stages of cell-plate formation were ob-tained and the first cell-plate membrane fusion factor, KNOLLE

was identified. Since then, our understanding of the 3D ultra-structure of cell-plate formation has been enhanced by the useof high-voltage EM tomography, and by the characterizationof many more cell-plate-associated factors through the use offorward and reverse genetics, which are aided by the com-pletion of the Arabidopsis genome sequence. The combi-nation of cell biological tools now available in plants is likely totest and render many of the hypotheses and questions raisedin this review obsolete within a very short time. Recent studieshave highlighted some of the dramatic similarities and differ-ences between animal, fungal, and plant cytokinesis. As moreinformation unfolds, the network of interactions becomesmore interconnected and complete through more genetic andbiochemical analyses. The complexity, similarity, anduniqueness of each organism’s and each cell type’s approachto the separation of one cell into two will show us just howmany ways there are to divide a cell.

Acknowledgments

We apologize to many authors for the omission of their references owingto restrictions in the length of the review and references numbers. Weare grateful to Dr David Rancour for discussion and critical reading of themanuscript. Research in the authors’ laboratory is supported by grantsfrom the Department of Energy, Division of Energy Biosciences (DE-FG02–99ER20332), from the National Science Foundation/Department of En-ergy/United States Department of Agriculture Collaborative Research inPlant Biology Program (BIR-9220331) and an award from the MilwaukeeFoundation.

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